Chunxin Wang

The Role of Mitochondria in Apoptosis

Mitochondria play key roles in activating apoptosis in mammalian cells. Bcl-2 family members regulate the release of proteins from the space between the mitochondrial inner and outer membrane that, once in the cytosol, activate caspase proteases that dismantle cells and signal efficient phagocytosis of cell corpses. Here we review the extensive literature on proteins released from the intermembrane space and consider genetic evidence for and against their roles in apoptosis activation. We also compare and contrast apoptosis pathways in Caenorhabditis elegans, Drosophila melanogaster, and mammals that indicate major mysteries remaining to be solved.

The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy

Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways.

Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL

Differential localization to the inner and outer mitochondrial membranes regulates PINK1 stability and function.

Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells

Localization of the dynamin-related GTPase Drp1 to mitochondria relies on the mitochondrial fission factor Mff.

High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Recent studies of the Parkinson’s disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.

Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria

Significance Selective autophagy of damaged mitochondria (mitophagy) requires protein kinases PINK1 and TBK1, ubiquitin ligase Parkin, and autophagy receptors such as OPTN, driving ubiquitin-labeled mitochondria into autophagosomes. Because all proteins have been genetically linked to either Parkinson’s disease (PINK1 and Parkin) or amyotrophic lateral sclerosis and frontotemporal lobar degeneration (TBK1 and OPTN), it is of great interest to understand their physiological functions. By utilizing quantitative proteomics we show that TBK1 phosphorylates four receptors on several autophagy-relevant sites. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and activation on mitochondria. TBK1-mediated phosphorylation of OPTN creates a signal amplification loop through combining recruitment and retention of OPTN/TBK1 on ubiquitinated mitochondria. Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagy-relevant sites, including the ubiquitin- and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria. TBK1 in turn phosphorylates OPTN’s UBAN domain at S473, thereby expanding the binding capacity of OPTN to diverse Ub chains. In combination with phosphorylation of S177 and S513, this posttranslational modification promotes recruitment and retention of OPTN/TBK1 on ubiquitinated, damaged mitochondria. Moreover, phosphorylation of OPTN on S473 enables binding to pS65 Ub chains and is also implicated in PINK1-driven and Parkin-independent mitophagy. Thus, TBK1-mediated phosphorylation of autophagy receptors creates a signal amplification loop operating in selective autophagy of damaged mitochondria.

Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy

Damaged mitochondria can be selectively eliminated by mitophagy. Although two gene products mutated in Parkinson’s disease, PINK1, and Parkin have been found to play a central role in triggering mitophagy in mammals, how the pre-autophagosomal isolation membrane selectively and accurately engulfs damaged mitochondria remains unclear. In this study, we demonstrate that TBC1D15, a mitochondrial Rab GTPase-activating protein (Rab-GAP), governs autophagosome biogenesis and morphology downstream of Parkin activation. To constrain autophagosome morphogenesis to that of the cargo, TBC1D15 inhibits Rab7 activity and associates with both the mitochondria through binding Fis1 and the isolation membrane through the interactions with LC3/GABARAP family members. Another TBC family member TBC1D17, also participates in mitophagy and forms homodimers and heterodimers with TBC1D15. These results demonstrate that TBC1D15 and TBC1D17 mediate proper autophagic encapsulation of mitochondria by regulating Rab7 activity at the interface between mitochondria and isolation membranes. DOI: http://dx.doi.org/10.7554/eLife.01612.001

Mutations in Fis1 disrupt orderly disposal of defective mitochondria

The mitochondrial fission protein Drp1 binds to Mff on mitochondria, followed by entry into a complex with Fis1 at the ER–mitochondrial interface. Mutations in Fis1 disrupt disposal of defective mitochondria when fission is induced by stress. Fis1 thus acts in sequence with Mff to couple mitochondrial fission with downstream degradation processes.

Bcl-2 family interaction with the mitochondrial morphogenesis machinery

The regulation of both mitochondrial dynamics and apoptosis is key for maintaining the health of a cell. Bcl-2 family proteins, central in apoptosis regulation, also have roles in the maintenance of the mitochondrial network. Here we report that Bax and Bak participate in the regulation of mitochondrial fusion in mouse embryonic fibroblasts, primary mouse neurons and human colon carcinoma cells. To assess how Bcl-2 family members may regulate mitochondrial morphogenesis, we determined the binding of a series of chimeras between Bcl-xL and Bax to the mitofusins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2). One chimera (containing helix 5 (H5) of Bax replacing H5 of Bcl-xL (Bcl-xL/Bax H5)) co-immunoprecipitated with Mfn1 and Mfn2 significantly better than either wild-type Bax or Bcl-xL. Expression of Bcl-xL/Bax H5 in cells reduced the mobility of Mfn1 and Mfn2 and colocalized with ectopic Mfn1 and Mfn2, as well as endogenous Mfn2 to a greater extent than wild-type Bax. Ultimately, Bcl-xL/Bax H5 induced substantial mitochondrial fragmentation in healthy cells. Therefore, we propose that Bcl-xL/Bax H5 disturbs mitochondrial morphology by binding and inhibiting Mfn1 and Mfn2 activity, supporting the hypothesis that Bcl-2 family members have the capacity to regulate mitochondrial morphology through binding to the mitofusins in healthy cells.

MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

The transcription factor TFEB is activated in a Parkin- and Atg5-dependent manner during mitophagy, and MiT/TFE transcription factor family members are required for the efficient clearance of damaged mitochondria.